Without limiting the scope of the invention, its background is described in connection with the concentration of proteins. The use of proteins and other polypeptides for therapeutics is on the rise in recent years as a way to expand and better treat patients since they are viewed to be less toxic and behave more predictably in vivo than other classes of drugs not naturally found in the body. Delivery of protein therapeutics has been limited primarily to dilute large volume intravenous injections to deliver the high dose required (100-1000 mg) and to avoid physical and chemical instabilities of proteins at high concentrations. A potentially less invasive method of administration is subcutaneous injection. Since the injection volume is limited to 1.5 ml, the concentration of the protein therapeutic is often substantially above 100 mg/ml. In addition to polypeptide stability, another major concern is the dramatic increase in viscosity for solution concentrations greater than 100 to 400 mg/ml due to protein interactions. If the primary interactions are attractive protein-protein interactions due to electrostatics, this increase in viscosity can been avoided by adding sodium chloride to increase the ionic strength of the solution and by varying the buffer species and pH of the solution. At these high concentrations, large excipient concentrations are often needed to protect against denaturation. An alternative approach would be to form a suspension of an insoluble protein in a non-aqueous solvent. The viscosity of highly concentrated suspensions can be much lower than for solutions and require smaller excipient levels to stabilize the protein. However, for successful delivery with concentrated suspensions, the particle size and suspension uniformity must be controlled in order to administer an accurate and uniform dose.
To date, there are relatively few examples of suspensions of proteins in non-aqueous media for medicinal purposes. Highly viscous suspensions of bovine somatotropin, marketed to increase milk production in dairy cows, and a bovine growth hormone releasing factor analog, used to release somatotropin from the cow's pituitary gland, are formulated in sesame oil and Miglyol oil, respectively. These viscous suspensions require a large 14-16 gauge needle for injection, whereas the preferred needle size for humans is between 25-gauge and 27-gauge. In addition, a few non-aqueous injections have been formulated as extended release formulations for the peptide insulin and very stable proteins such as protein C and a proprietary monoclonal antibody with the aid of viscosity enhancers and gel forming polymers in the presence of diluents such as benzyl benzoate or benzyl alcohol. However, these formulations are syringeable only with a larger 21-gauge needle causing considerable pain upon injection leading to non-compliance and the high levels of excipients reduce the overall concentration of the protein in the formulation. Another option is to crystallize the protein or monoclonal antibody and form an aqueous suspension of the crystals. This approach has been shown for three monoclonal antibodies and insulin. However, crystallization of high molecular weight proteins can be very difficult due to the high degree of segmental flexibility, and is more feasible for small peptides that have a much lower degree of flexibility.